The present invention relates to detection of micro-organisms, particularly to the identification organisms by fingerprinting biochemicals and DNA fragments, and more particularly to an inexpansive, small multiple channel PCR/CE instrumentation for rapidly identifying micro-organisms and DNA fragments.
The detection of micro-organisms in the field will be an essential element of any future military action. The identification of threat organisms by fingerprinting biochemicals and DNA fragments has matured to a point that in the laboratory specific identifications can be made. However, the laboratory instrumentation is not readily adapted for field applications.
The analysis of DNA fragments by electrophoresis is well established and essential for all types of genetic and biochemical studies. A thin electrophoresis gel is prepared, poured, and a potential of 100-300 volts (20-100 milliamps) applied. Separation of DNA fragments can take from 1-8 hours, depending upon the resolution desired and hardness of the gel. Preparing the gels, adding samples to each electrophoresis lane, separating, staining, and identifying DNA fragments visually is time consuming.
Standard DNA analysis methods sometime require hundreds of samples to be analyzed simultaneously. After electrophoretic separation, the gel must be stained and photographed in order to generate a permanent record which requires manipulation of the gel material and potential exposure to toxic staining chemicals. The electrophoric gels are prepared either from agarose or polyacrylamide and they are typically used only once. These water based gels will not store well without drying out and deteriorating within a few hours. Thus, these gels are not suitable for field use.
Although gel electrophoresis has been the cornerstone of genetic research, the current analytical technique is labor intensive, slow, and difficult to perform in a fully automated manner. What is needed is a multisample analysis device which can: 1) rapidly screen samples, 2) requires minimum sample preparation, 3) has sensitive and precise separation capabilities, 4) is attractive to researchers already performing standard gel electrophoresis, and 5) can be easily and quickly used in the field.
Capillary electrophoresis (CE) is one approach which has become increasingly more popular in the last few years, and more commercial CE systems are now available. CE is fast, accurate, highly specific, and requires only minimum (mL) amounts of sample. Small, 50-100 micron internal diameter (i.d.) and 10-20 cm long, capillary tubes perform exceptionally well and typically have 100,000-800,000 theoretical-plate separation characteristics. Because of the small capillary size, the heat dissipation of the column is very good and thermal peak broadening is almost nonexistent. Although, performing high resolution separations of DNA bands within minutes, commercial CE instruments are configured only with a single column.
In addition to analyzing only one sample at a time, an interface between polymerase chain reaction (PCR) sample preparation and commercial capillary electrophoresis instruments does not exist. PCR samples must be manually transferred to the CE instrument for analysis. This is very time consuming and difficult when only a limited amount of sample is available. Although CE is faster than gel electrophoresis, a CE system which analyzes many samples in parallel does not exist and no methods are now available to directly and immediately analyze the products of the PCR reactions.
The present invention provides a solution to the above-mentioned drawbacks of the existing analysis systems and provides multiple channel integrated PCR/CE instrumentation to rapidly identify micro-organisms and DNA fragments. The instrumentation provided by this invention enables field use and is of a "throw-away" nature, thus minimizing cross contamination and the potential for false positive identification between samples, as well as being designed for multiple users with individual applications. The integral PCR/CE instrument can be manufactured from plastic and eliminates the complicated electronic hardware of previous instruments, while being compact (hand-held). The instrument is small and compact so that 100 PCR/CE reactions/analyses can be performed easily on one detection device.